Intra-mammary teat sealant formulation and method of using same to reduce or eliminate visual defects in aged cheeses

ABSTRACT

Described is an intra-mammary teat sealant and a corresponding method of forming a physical barrier in the teat canal of a non-human animal for prophylactic treatment of mammary disorders during the animal&#39;s dry period. The method includes the step of infusing a teat seal formulation including a metal salt in a glyceride-containing gel base into the teat canal of the animal. The method also prevents the formation of black spot defect in dairy products, especially cheddar cheese, made from the milk of animals so treated.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is claimed to provisional application Ser. No. 61/207,879,filed Apr. 8, 2009, the content of which is incorporated herein byreference. This application is also related to co-pending applicationSer. No. 11/869,966, filed Oct. 10, 2007, which is incorporated hereinby reference.

FIELD OF THE INVENTION

The invention is directed to a metal-containing, intra-mammary teatsealant to prevent mastitis in dry cows. The intra-mammary teat sealantdoes not cause visual defects in dairy foods (especially cheese) madefrom milk from treated animals. The invention is further directed to amethod to prevent “black spot defect” (BSD) in cheese.

BACKGROUND

Mastitis in dairy herds is one of the most costly and difficult diseasesencountered by dairy producers. Conventional therapies aimed at curingclinical mastitis include intra-mammary antimicrobial therapy. Despitethe commercial availability of numerous intra-mammary antimicrobialproducts, cure rates for clinical mastitis remain perplexingly low: 46%for Streptococcus spp., 21% for Staphylococcus spp., and only 9% forStaphylococcus aureus mastitis. See Wilson et al. (1996) NationalMastitis Council Proceedings 164-165, and Crandall et al. (2005) NMCAnnual Meeting Proceedings 215-216. Thus, dairy producers often managethe disease simply by culling mastitis-prone animals from their herds.

Because of the difficulty in treating mastitis, prevention of newintra-mammary infections is a major focus in the dairy industry. Therate of new infections is significantly higher during the dry period ascompared to new infections during the lactating period. (For example,one study showed that 61% of all new gram-negative intra-mammaryinfections occurred during the dry period. See Todhunter et al. (1995)J. Dairy Sci. 78:2366.) The three-week period immediately following dryoff, and the two weeks prior to calving, are periods particularly proneto new infections. Thus, in recent years dairy producers have focused aconsiderable amount of effort in “preventive maintenance” of cows duringtheir dry period.

In April of 2003, an internal (or “intra-mammary”) teat sealant (ITS)for use in dry cows was introduced in the US market. Marketed in the USunder the “ORBESEAL” trademark (US Trademark Registration Nos. 2,772,198and 3,120,693), the product was developed in New Zealand. The“ORBESEAL”-brand ITS introduced into the US market contains 65% w/wbismuth sub-nitrate dispersed in a viscous paste. The ITS product doesnot contain any antibiotics, nor does the product contain any activeantimicrobial agents. The ITS is injected into the teat end using atubular applicator syringe, in the same fashion as applying a dry cowantibiotic. The ITS product fills the fissures and folds of the teatcanal, thereby creating a physical barrier to pathogens. See U.S. Pat.No. 6,254,881, issued Jul. 3, 2001, incorporated herein by reference.

Initial studies of the “ORBESEAL”-brand product in New Zealand concludedthat the product functioned as well as a broad spectrum, long-actingintra-mammary antibiotic in preventing new intra-mammary infections atcalving and in preventing clinical appearance of mastitis through thefirst five (5) months of lactation. See Woolford et al. (1998) NewZealand Veterinary Journal 46:1. A more recent study in the US alsoconcluded that this ITS product improved the udder health of cowsalready infused with cloxacillin benzathine. See Godden et al. (2003) J.Dairy Sci. 86:3899-3911. Thus, the “ORBESEAL”-brand ITS has proven to bean effective tool in reducing the number of new cases of mastitis indairy cows during their dry period. Despite its relatively recentintroduction into the US market, the “ORBESEAL”-brand product hasenjoyed widespread market acceptance and is used extensively in US dairyherds. In short, the “ORBESEAL”-branded product is very good for itsintended purpose of preventing mastitis.

Subsequent to the introduction of the “ORBESEAL”-brand ITS product inthe US, a visual defect in aged dairy products, most notably agedcheddar cheeses, began to appear. The visual defect takes the form ofsmall, black spots (roughly 0.5 to 5 mm in diameter) that appearthroughout the aged cheese. The spots are a purely aesthetic, visualdefect that lowers the graded quality (and hence the market value) ofthe cheese affected with the problem. The spots are not accompanied byany organoleptic defect in the cheese. Cheese affected with the blackspots is saleable, albeit at a lower grade than unaffected cheeses. Thedefect has been termed “black spot defect” (BSD).

SUMMARY OF THE INVENTION

A first version of the invention is directed to a method of forming aphysical barrier in the teat canal of a non-human animal forprophylactic treatment of mammary disorders during the animal's dryperiod and simultaneously preventing BSD in dairy products made with theanimal's milk. The method comprises infusing a teat seal formulationinto the teat canal of the animal. The teat seal formulation may containbismuth or may be bismuth-free. The formulation is administered in anamount sufficient to form a physical barrier to entry of microorganismsinto the teat, but does not cause black spot defect in diary productsmade with milk from the animal. Preferably, the teat sealant is devoidof anti-infective agents (i.e., the teat sealant preferably does notcontain antibiotics or other anti-infective active agents). Preferablythe method comprises infusing a teat seal formulation comprising atleast about 30% by weight of a non-toxic metal salt, more preferablyabout 50% to about 75% by weight of the non-toxic metal salt, and morepreferably still about 65% by weight of the non-toxic metal salt. Thepurpose of the salt is primarily to impart sufficient density to thecomposition so that the ITS “settles” into the teat canal.

In one version of the method, the non-toxic metal salt is selected fromthe group consisting of bismuth salts, titanium salts, zinc salts,barium salts, and combinations of these salts. The most preferred saltsare bismuth sub-nitrate, titanium dioxide, zinc oxide, barium sulfateand combinations of these salts. Other non-toxic salts of these metalare explicitly within the scope of the invention, such as halides,sulfates, phosphates, carbonates, nitrates, sulphamates, acetates,citrates, lactates, tartrates, malonates, oxalates, salicylates,propionates, succinates, fumarates, maleates, and the like.

The gel base can be any suitable gel formulation, a host of which areknown in the pharmaceutical arts. The preferred gel base comprisesmono-, di-, and/or triglycerides, which give vastly improvedcleanability. The glycerides may contain identical side-chains ordifferent side-chains, which may be derived from saturated orunsaturated fatty acids. Thus, by way of non-limiting example, themono-, di-, and/or triglycerides may comprise side-chains derived fromdecanoic acid, lauric acid, myristic acid, palmitic acid, pentadecanoicacid, stearic acid, arachidic acid, behenic acid, lignoceric acid,margaric acid, myristoleic acid, palmitoleic acid, oleic acid, gadoleicacid, erucic acid, ricinoleic acid, linoleic acid, linolenic acid,licanic acid, margaroleic acid, arachidonic acid clupanadonic acid,eicosapentaenoic acid, docosahexaenoic acid, and the like. Glycerideshaving longer or shorter side-chains may also be used. The glyceridesmay comprise, or be derived from, a natural animal or vegetable oil orfat, such as (but not limited to) animal fats, canola oil, coconut oil,corn oil, cottonseed oil, linseed oil, palm oil, palm kernel oil,rapeseed oil, soya bean oil, sunflower oil, fish oil, algae oil, and thelike. Particularly preferred gel bases are canola oil, corn oil, and/orcottonseed oil. There are a host of commercial suppliers of glycerides,such as ABITEC Corporation, Columbus, Ohio. ABITEC's product “CAPTEX355,” catalog nos. 65381-09-1 and 73398-61-5, a caprylic acidtriglyceride, works very well in the present invention.

Thus, in its most preferred form, the intra-mammary teat sealantcomprises, in combination, a gel base comprising a glyceride; and anon-toxic metal salt dispersed in the gel base. The preferredcorresponding method thus comprises infusing an amount of a teat sealformulation into the teat canal of the animal. The teat seal formulationcomprises a non-toxic metal salt dispersed in a gel phase comprising aglyceride, wherein the amount of the teat seal formulation infused issufficient to form a physical barrier to entry of microorganisms intothe teat canal. The teat seal formulation also does not cause black spotdefect in diary products made with milk from the animal.

Another version of the invention is directed to an intra-mammary teatsealant consisting essentially of a gel base including mono-, di-,and/or triglycerides, in combination with a non-toxic metal saltdispersed in the gel base, wherein the metal salt is bismuthsub-nitrate. As noted earlier, the teat sealant preferably comprises atleast about 30% by weight, more preferably about 50% to about 75% byweight, and more preferably still about 65% by weight of the metal salt.

Yet another version of the invention is an improvement to intra-mammaryteat sealants. Specifically, in a method of forming a physical barrierin the teat canal of a non-human animal for prophylactic treatment ofmammary disorders during the animal's dry period, wherein the methodcomprises the step of infusing a seal formulation into the teat canal ofthe animal without an anti-infective agent, the improvement of thepresent invention comprises infusing a teat seal formulation comprisinga non-toxic metal salt(s) in a glyceride-containing gel base. Theimprovement prevents the formation of black spot defect in dairyproducts made from the milk of treated animals.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, 5, 6,from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations of the presentinvention shall include the corresponding plural characteristic orlimitation, and vice-versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The methods of the present invention can comprise, consist of, orconsist essentially of the essential elements and limitations of themethods and products described herein, as well as any additional oroptional ingredients, components, or limitations described herein orotherwise useful in synthetic organic chemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photographs of a typical black spot defect in an 18kg block of aged white cheddar cheese. FIG. 1A shows the surface of theblock, with a black spot defect readily visible. FIG. 1B is a magnifiedview (with a superimposed ruler) showing the dimensions of the defect.Such spots are equally distributed throughout the cheese.

FIG. 2 is an electron photomicrograph of a black spot defect exhibitingcharacteristic hair- or rod-like structures of bismuth (III) sulfidenanorods. No such structures appeared in any non-BSD cheeses or non-BSDcheese regions tested. The rods shown in FIG. 2 have diameters rangingfrom 37.09 nm to 129.33 nm.

FIG. 3 is an electron photomicrograph of a single rod-like structurefrom a black spot defect region. The rod shown in 130.88 nm in diameterand exhibits a line bisecting the length of the rod.

FIGS. 4A and 4B are images of laboratory-induced black spot defects. InFIG. 4A, the various components of “ORBESEAL”-brand intra-mammary teatsealant (ITS) were blended with cheese and each spot was photographedimmediately. FIG. 4B shows the same spots photographed after exposureeither to volatiles from aged cheddar cheese or hydrogen sulfide gas.Sites 4 and 5 contain bismuth subnitrate and the intact “ORBESEAL”-brandITS formulation, respectively.

FIG. 5 is a histogram depicting the cleanability of various types ofITS's.

FIG. 6 is a graph depicting viscosity of the ITS formulations atdifferent temperatures. X-axis depicts temperature in ° C., Y-axisdepicts viscosity.

DETAILED DESCRIPTION OF THE INVENTION

Starting in late 2003, a number of inquiries have been made by cheesemakers to the University of Wisconsin-Madison, Department of FoodScience and Center for Dairy Research (CDR), seeking information aboutthe appearance of a novel “black spot defect” in aged cheeses, notablyaged cheddar cheese. Historically, grey to black discolorations incheese have been the result of several different and distinct causes,including the growth of specific microorganisms (e.g., certainenvironmental propionibacteria or molds) or the contamination of cheesewith food-grade lubricant debris. The particular BSD noted by the cheesemakers, however, did not fit the profile of a bacterial contaminant orother spoilage organism, nor did it appear to be lubricant debris thathad found its way into the milk stream or other debris introduced duringthe cheese-making process.

Thus, the first step was to determine the chemical structure of theblack spot defect. A great deal of effort was initially made to extractthe affected regions of BSD cheese. Extraction efforts using a widespectrum of organic solvents of varying polarity, hydrophobicity, etc.,proved fruitless as a means of isolating any type of organic pigmentmass from the cheese matrix. Although extraction of the BSD with organicsolvents was not successful, the extraction efforts did yield usefuldata. Notably, because the pigment did not dissolve or diffuse into suchsolvents, it could be concluded (with a high degree of probability) thatthe black spot pigment likewise would not dissolve or diffuse within thecheese matrix itself.

Visual examination of a growing number of cheese samples exhibiting thedefect (samples accumulated from commercial cheese makers) confirmedthis conclusion—the black spot pigment is well contained and does notappear to diffuse into the cheese matrix. See FIGS. 1A and 1B, which arephotographs of a typical 18 kg block of aged, white cheddar cheeseaffected with the BSD. FIG. 1A is a photograph of the outer surface ofthe cheese block. FIG. 1B is a magnified view of a single black spot,with a ruler superimposed on the image to show the dimensions of thespot. Spots such as the one shown in FIGS. 1A and 1B typically areequally distributed throughout the cheese block, ranging in size from <1mm to about 5 mm in diameter. The frequency of the spots within anygiven 18 kg cheese block varies widely, from <10 per block to well over100.

There were some anecdotal reports received from cheese makers thatspecific aging and storage strategies might aid in dissolving ordiffusing the spots to the point that they are no longer visuallynoticeable. (Because the defect is not accompanied by any organolepticdeficiency, “fading” the spots would ameliorate the condition.) Such aneffect, however, is highly unlikely given the stability of the pigmentto the organic solvents employed in the extraction efforts. In short,the extraction experiments performed by the present inventor usedsolvents having hydrophobicities similar to milk fat. If the black spotpigment dissolved or diffused into the cheese matrix itself (via anaging or storage protocol), the pigment should likewise readily dissolveor diffuse into an organic solvent having physical characteristicssimilar to milk fat. That result did not occur in the lab. Moreover,given the typical pH/acidic environment in cheese, and the typicalaging/shelf life periods associated with most aged cheddar-type cheeses(0.5 to 2 years), the anecdotal evidence that the defect can beameliorated via aging or storage protocols is without merit.

One experiment, however, proved most enlightening: the black spotpigment is readily dissolved in nitric acid. This strongly suggestedthat the pigment was an inorganic salt. Coupled with the timing of thefirst appearance of the defect, a working hypothesis was formulated,namely that the ITS was either a causative agent of (or at leastcorrelated with) the BSD. The discovery that the black spot pigmentreadily dissolves in acid supported a further hypothesis that thepigment may be comprised of bismuth III sulfide. Thus, it was concludedthe “ORBESEAL”-brand product, which in the US contains 65% by weight ofa bismuth-containing salt, was likely being inadvertently introducedinto the milk stream. As noted above, the “ORBESEAL”-brand product hasbeen commercially successful because it forms a tight physically barrierto the entry of pathogens into the teat canal. However, removing theproduct from a treated animal requires stripping of the animal's teats.It appeared that some of the ITS remained in the teats after strippingand was finding its way into the cheese milk.

The next phase of research operated pursuant to a hypothesis thatbismuth III sulfide was in fact the causative agent of the BSD. Bismuthsubnitrate itself is white and relatively chemically inert. Thus, itstrace presence in fluid milk, mozzarella cheese, and yogurt is notreadily apparent visually. However, in aged cheeses with high flavorintensity, the black spot defect appears prominently. Thus, it washypothesized that bismuth III sulfide (a black, relatively insolublesalt) was the product of a reaction between bismuth subnitrate (from theITS) and hydrogen sulfide produced within the aging cheese by theactions of ripening microflora, enzymes, and certain cofactors acting onthe protein/amino acid components of cheese.

In short, the hypothesis was that bismuth subnitrate made its way intothe milk stream due to incomplete removal of the ITS prior to milking.The bismuth subnitrate then reacted with hydrogen sulfide to yieldbismuth III sulfide according to Equation 1

4BiNO₃(OH)₂BiO(OH)+H₂S→Bi₂S₃ (insol., black)  (Eq. 1)

The product, bismuth Ill sulfide (or simply bismuth sulfide) is arelatively insoluble, black salt.

In addition to having a specific elemental target, bismuth, it washypothesized that, under the conditions or chemical environment presentwithin the cheese matrix, the Bi₂S₃ molecules would form a crystallinestructure referred to in the literature as nanorods or nanowhiskers. SeeW. Zhang et al. (2001) Sol. State Comm. 119:143-146 and B. Zhang et al.(2006) J. Phys. Chem. 110:8978-8985. These bismuth-containing nanorodswould thus constitute light-diffracting particles capable of impartingthe grey to black hue seen in the black spot defect.

Efforts were then focused on confirming: 1) the elemental presence ofbismuth in the black spot defects; and 2) confirming the physicalpresence of bismuth Ill sulfide nanorod structures within the black spotdefects.

Confirming the presence of bismuth within the black spots wasinvestigated using inductively coupled plasma mass spectroscopy (ICPMS).AOAC International (Association of Analytical Communities) method 993.14was used. The first efforts screened multiple black spots for thepresence of several elements that could be contributing to BSD. Theinitial experiments focused on metal salts/oxides typical of those foundin milk- and cheese-handling/conveying equipment, and other residualmetal derivatives present in food-grade processing. As a measure ofcontrol, cheese compositional analyses were conducted. Specifically,protein, ash, and moisture were measured using methods 2001.14, 935.42,and 926.08, of the Official Methods of Analysis, AOAC 17^(th) Edition,respectively (copyright 2000, ISBN: 0935584-67-6). Fat was measuredaccording to the method described in the Official Methods of Analysis,AOAC 17th Edition.

Transmission electron microscopy (TEM) studies were performed asfollows: approximately 100 μl double-distilled water was added tosamples and the mixture was pulverized into a suspension with a glassrod. Approximately 5 μl aliquots of suspended sample were deposited ontopolyvinyl alcohol-formaldehyde acetal-coated 300 mesh copper TEM grids(Ted Pella, Inc., Redding, Calif.). Excess sample was wicked away withsmall sections of filter paper and the remaining sample was dried to thesurface of the grid at room temperature. In some cases, NANO-W-brand TEMnegative stain (Nanoprobes, Incorporated, Yaphank, N.Y.) was appliedover the dried sample to enhance contrast and visibility. Specimens wereobserved with a Philips CM 120 electron microscope and images werecollected with a MegaView 3 Digital camera (from SIS, Ringoes, N.J.).Measurements were taken with SIS-brand analysis software (Ringoes, N.J.)calibrated with reference samples of known lengths.

ICPMS results demonstrated the presence of the elements chromium,copper, iron, nickel, and bismuth in the BSD region. Althoughincremental increases in the elements chromium, copper, iron, and nickelwere found, bismuth concentrations in the BSD region were routinelythree orders of magnitude greater than the same cheese assayed innon-BSD areas. These results show that bismuth is the only elementpresent in sufficient quantities to participate in a pigment-generatingreaction.

Several hundred TEM images of BSD regions of cheese samples werecaptured with a single, consistent conclusion. Nanorods typical of thosereported in the literature cited above were uniquely present in the BSDcheese region. An example of such an image is presented in FIG. 2. Thenanorod structures shown in FIG. 2 are too small to be readily detectedwith a light microscope. The nanorods shown in FIG. 2 range in diameterfrom about 69 nm to about 130 nm. The nanorods are very stable to thepotentially abusive conditions of TEM. The rods appear to have aslightly mottled surface and they exhibit a characteristic line runningthe length of the nanorod. See FIG. 3, which is an increasedmagnification view of a single nanorod. The presence of such structuresis consistent with the presence of bismuth sulfide nanorods formed underthe conditions present in the cheese matrix.

To confirm the reactivity of bismuth subnitrate as a reactant in formingbismuth Ill sulfide nanorods, additional assays were conducted to see ifBSD could be purposefully recreated in the lab. In short, cheeses weremanufactured with known amounts of ITS components and subjected toeither the authentic volatile gasses produced by maturing cheeses orexposed directly to the hypothesized bismuth subnitrate co-reactant,hydrogen sulfide gas. In both situations, the responses were invariablythe same: When cheese samples containing bismuth subnitrate or thecomplete ITS formulation were exposed to authentic cheese volatiles orto “chemical standard”-grade H₂S gas, each formed identical blackpigmentation with the accompanying presence of nano-rod structures,further confirming that bismuth subnitrate is the culprit in BSD. Theresults are shown in FIG. 4. No other ITS component formed black spotswhen so exposed. Furthermore, the susceptible sites formed identicallyblack pigmentation when exposed to either authentic cheese volatiles orto hydrogen sulfide gas, thus confirming that hydrogen sulfide gas wasthe suspected co-reactant.

From a cheese manufacturing and aging or ripening standpoint, it is notreasonable to consider targeting the elimination of hydrogen sulfide gasproduction as a means of controlling BSD. Hydrogen sulfide is a highlyaroma active compound, the product of microbial, enzymatic and co-factoractivities against sulfur-containing amino acids such as cysteine. SeeArfi et al., (2002) Appl. Microbiol. Biotechnol. 58:503-510. There isample research to support the claim that hydrogen sulfide gas is anecessary and/or valued component of typical aged cheddar cheese flavor.See Burbank & Qian (2005) J. Chrom. 1066:149-157. Even if a scheme wasdevised to eliminate the production of hydrogen sulfide (by interruptingof dozens of complex metabolic pathways) the resulting final productruns the risk of a flavor character unacceptable to cheese graders andconsumers.

EXAMPLES

The following Examples are included solely to provide a more completedescription of the invention disclosed and claimed herein. The Examplesdo not limit the invention in any way.

Example 1

A test ITS using a combination of zinc oxide and titanium oxide as themetal salts was formulated. The test ITS was identical to the“ORBESEAL”-brand formulation, with the exception that it did not containany bismuth or bismuth-containing salts. The test ITS comprised zincoxide, titanium dioxide, mineral oil (30-40%), and aluminium stearate.

To prepare a batch of ITS, liquid paraffin (e.g., mineral oil) isdelivered into a suitable vessel equipped with a mixer. Aluminumstearate is added and the mixture is stirred and heated to about 160° C.until homogeneous (about two hours).

The non-toxic, non-bismuth containing salt is then added in portions tothe mixture, with stirring, until the desired amount of metal salt hasbeen added. The mixture is then stirred until homogenous. The productsis then transferred into conventional injector tubes for intra-teatadministration.

Example 2

The object of this Example was to compare retention within the teats ofnon-lactating dairy cows of an ITS that did not include bismuthsub-nitrate as compared to the “ORBESEAL”-brand product.

The study was performed at the Blaine Dairy of the University ofWisconsin-Madison (UW), in Arlington, Wis. Sixteen (16) cows (n=64teats) were enrolled on the day of dry off. All enrolled cows wererequired to have four functional quarters and no visible sign ofmastitis. All cows were dried off and received intramammary antibioticdry cow therapy (DCT) according to standard UW dairy herd protocols.Parity and milk yield (at dry off) were recorded for each cow. Uponinitial enrolment, teats were scored for shape, length, diameter anddegree of teat end hyperkeratosis. Within each cow, two teats wereassigned to receive the “ORBESEAL”-brand ITS and two teats were assignedto receive the test ITS. The administration protocol was designed toensure that each product was administered uniformly among teatlocations, eight teats each per product administered in each location(right-rear, right-front, left-rear, left-front). Sealant tubes wereweighed before and after administration to determine the net volumeadministered. Prior to receiving DCT & the internal teat sealant, teatends were cleaned using a single 70% isopropanol alcohol wipe andpartial insertion technique was used to reduce the probability ofintroducing teat skin pathogens. After administration of the internalsealant, teats were dipped with an external teat disinfectant.

Teats were examined on Days 1, 2, 3, 4, 5, 6, 7, 14, 28, 42 and atcalving to detect redness, swelling and/or sealant leakage. On Days 14,28, 42 and at calving, sealant was removed from one teat (eight teatsfor each sealant per removal day) of each cow by hand stripping. Theremoved sealant was collected with the first milk into graduated 50 mlplastic vials. The vials were centrifuged (3000 rpm×5-7 minutes), thesupernatant rinsed, and the recovered sealant weighed. The amount ofrecovered sealant was compared at each period between the testITS-treated teats and the “ORBESEAL”-brand ITS-treated teats. Follow upsamples were collected at Day 1 post-calving using the same procedure.At all sampling periods, after removal of the sealant, teats were dippedwith an external teat disinfectant. After calving, quarter milk sampleswere aseptically collected from all quarters and cultured to identifyintramammary infections.

Group Characteristics—Teat Length and Volume:

A total of 16 cows were enrolled into the study for a total number of 64teats; 32 teats received the test ITS and 32 teats received the“ORBESEAL”-brand ITS. The teat length and volume for the test populationis shown in Table 1:

TABLE 1 Mean, Standard Deviation, and Standard Errors for Teat Lengthand Volume by Compound Group A Group B (“ORBESEAL”-ITS) (Test ITS) NMean S.D. S.E. N Mean S.D. S.E. p Length 32 5.12 0.88 0.16 32 5.11 0.860.15 0.95 Volume 32 23.60 8.61 1.52 32 25.72 9.73 1.73 0.36

There was no significant difference in teat length or volume for teatsin Group A or Group B (p>0.36). Overall teat length was 5.11 cm, rangingfrom 3.3 cm to 7.3 cm. The average teat length was 5.12 cm for Group Aand 5.11 cm for Group B, and ranged from 3.3 cm to 7.3 cm for Group Aand from 3.5 cm to 7.10 cm for Group B. A two Sample paired t-test wasperformed to test the null hypothesis that the mean teat length in thetwo treatment group did not differ. There was no significant differencein teat length between teats randomized to receive either product(p=0.95).

The overall teat volume was 24.66 cm³, ranging from 12.47 cm³ to 54.34cm³ (std. dev. 9.17 cm³). The mean teat volume was 23.6 cm³, rangingfrom 12.54 cm³ to 54.34 cm³ (std. dev. 8.60 cm³) in Group A. In Group B,mean teat volume was 25.71 cm³, ranging from 12.47 cm³ to 48.25 cm³(std. dev. 9.73 cm³). A two sample paired t-test was used to test thenull hypothesis that the teat volume in the two groups did not differ.There was no significant difference in teat volume between teatsrandomized to receive either product (p=0.95 and p=0.35; log transformedanalysis).

Hyperkeratosis: Teat-end health was scored for hyperkeratosis using thefollowing scale: No ring (N), Smooth Ring (S), Rough (R), Very Rough(VR). The distribution of teat scores was: N (n=21; 32.8%), S (n=31;48.4%), R (n=11; 17.2%) and VR (n=1; 2%). An X² test confirmed that thedistribution of hyperkeratosis score was not associated with treatmentgroup (p=0.13).

TABLE 2 Descriptive Statistics for Hyperkeratosis Group A (“ORBESEAL”Group B ITS) (Test ITS) Overall Score Frequency Percent FrequencyPercent Frequency Percent N 13 40.63 8 25.00 21 32.81 S 11 34.38 2062.50 31 48.88 R 7 12.88 4 12.50 11 17.19 VR 1 3.23 n/a n/a 1 1.56

Amount of Sealant Administered, Recovered and Lost: Statistical analysesusing a paired t-test were performed to determine if the amount ofsealant administered, recovered, or lost (not recovered) did not differbased on treatment group.

TABLE 3 Two Sample Paired t-Test for the Mean of Administered, Recoveredand Lost Sealant by Product Group A (“ORBESEAL” Group B ITS) (Test ITS)Overall N Mean S.D. N Mean S.D. N Mean S.D. P Administered 32 3.46 0.8532 3.77 0.96 64 3.62 0.91 0.12 Recovered 32 0.85 1.42 32 0.79 1.57 640.82 1.49 0.89 Lost 32 2.71 1.37 32 3.04 1.55 64 2.88 1.46 0.40

Of the four (4) grams in each tube, the overall amount of sealantadministered was 3.62 grams. There was no significant difference in theamount of “ORBESEAL”-brand ITS (3.46 gram) or test ITS (3.62)administered (P=0.12).

Overall, the amount of sealant recovered was 0.82 gram and there were nosignificant differences based on treatment (P=0.89). The overall amountof sealant lost was 2.88 grams and did not differ by treatment group(P=0.40). The amount of sealant recovered tended to be associated withrecovery date (P=0.08) with more sealant recovered on day 14 as comparedto other recovery periods (day 14, recovery=1.6 grams; day 28recovery=0.68 grams; day 42 recovery=0.65 grams; calving recovery=0.33grams).

Simple linear regression was used to determine that there was nosignificant relationship between the amount of administered sealant andteat volume (p=0.59, p=0.53).

For the recovered sealant a simple linear regression test was performedto test the null hypothesis that there was no significant linearrelationship between the amount of recovered sealant and the teatvolume. Only 6% of the recovered sealant was accounted for by teatvolume (P=0.05).

The proportion of administered sealant was not significantly associatedwith teat volume, while the recovered sealant was correlatedsignificantly with the teat volume but only for a small proportion (6%).

A one-way ANOVA was used to determine univariate relationships betweenthe amount of administered and recovered sealant and the teat position,the product, the cow.

TABLE 4 Administered and Recovered Volume Univariate Association Table(P values) Administered Recovered (p) (p) Teat volume 0.53 0.13 Teatposition 0.56 0.52 Product 0.19 0.88 Day — 0.07 Cow 0.05 0.17

Example 3

The object of this Example was to develop an ITS with improvedcleanability properties. In this Example, an ITS similar to the onedescribed in Example 1 was made, but the mineral oil gel was replacedwith a triglyceride-based mobile phase. These sealants, which arepreferred, proved to have good barrier properties, as well as improvedcleanability properties using conventional teat-cleaning protocols (ascompared to mineral oil-based sealants). Milk producers have found thatit is extremely difficult to remove residual from conventional, mineraloil-based ITS formulations from milk contact surfaces using standardcleaned-in-place (CIP) procedures. Therefore, an ITS which has goodbarrier properties and which is also easy to clean from surfaces thatcontact milk is very desirable in the market.

Due to various factors, many milk producers do not adequately remove ITSfrom the udder before milking. It is unlikely that this outcome can beeliminated through training and education efforts. Furthermore, manymilk producers do not routinely achieve the recommended temperature andcleaning compound concentration requirements during clean up, thusexacerbating the build-up of residue from conventional ITS formulationson milking equipment. Dairy producers' cleaning practices are not likelyto change through education efforts. However, a viable approach toimproving the cleanability of residual ITS resides lies in altering thechemistry of the ITS product itself.

The salt component of the ITS, whether it be bismuth sub-nitrate, zincoxide or some other salt, is not influenced or solubilized to any greatdegree using standard cleaning protocols. The salts themselves aredispersed particulate matter. The salts are not soluble in either thecontinuous gel phase (conventionally mineral oil), nor in the aqueouscleaning phase. Further still, the mineral oil component (a viscousmixture of alkanes) of an ITS product is essentially non-reactive tostandard cleaning chemicals (e.g. chlorinated alkaline cleaner). Mineraloil is also sufficiently non-polar to resist solubilization by aqueouscleaning solution. In short, once an ITS comprising a mineral oil gelphase is deposited onto a milk contact surface, it is extremelydifficult to remove the ITS using standard cleaning protocols.

The role that mineral oil plays in an ITS formulation is as an inertcontinuous phase dispersant. The present inventor has discovered thatthis role can also be played by materials such as a mono-, di-, and/ortriglyceride oil. The benefit of replacing mineral oil with, forexample, a triglyceride material, is that unlike mineral oil,triglyceride is reactive to the most common milk equipment cleaningagent, alkaline cleaner (i.e., sodium hydroxide). Fatty acids esterifiedas glycerides are chemically converted at high pH with sodium hydroxideinto their corresponding sodium salts, e.g. sodium stearate, and arereadily removed from surfaces and dispersed into the aqueous phase ofthe cleaning solution. Triglyceride oils such as corn, cottonseed orcanola oil are readily available, are heat stable (for sterilizationpurposes), and can be altered to have different stability and meltingproperties.

Glyceride-based ITS's compare very favorably with mineral oil-basedITS's in terms of barrier properties. The triglyceride-based ITS's alsoexhibit at least a 10-fold improvement in cleanability as compared tomineral oil-based ITS's. Because of the improved cleanability, atriglyceride-based ITS may utilize a bismuth-containing salt, such asbismuth sub-nitrate as the salt component.

To test ITS's made from a glyceride gel base, a small clean-in-place(CIP) loop was constructed. The CIP loop comprised an 11-gallon tank,flexible sanitary lines, a centrifugal pump, a throttling valve, and adigital flow meter. For test surfaces, 2″×2″ coupons of 316 stainlesssteel and plastic were used. The cleaned coupons were weighed. Samplesof known weight of ITS were then applied to the surface of each couponand the coupons weighed again. The coupons were then washed in the CIPloop. Each coupon was inserted into the end of one of the flexible linesof the CIP lop and held in place with a hose clamp. The pump was thenturned on and the flow rate modulated with the throttling valve toachieve the desired target flow rate as displayed on the digital flowmeter. A digital thermocouple was used to monitor the temperature. Withthe coupon and ITS treatment in place, the system was filled with hotwater at 130° F., the pump turned on and adjusted for flow. One (1) ozof chlorinated alkaline cleaner per gallon of water in the CIP loop wasadded and the system was allowed to operate for 10 minutes. The couponwas removed, dried and weighed to determine the amount of ITS removed.

The results demonstrate that there are no unique differences incleanability between the “ORBESEAL”-brand ITS and the zinc-based ITSdescribed in Example 1, both of which use mineral oil as the gel phase.See Table 5, shown in FIG. 5, n=3. Table 5 depicts the weight percentremoval of ITS treatments from stainless steel and plastic coupons. Key:ss=stainless steel surface; pl=plastic surface; orb=“ORBESEAL”-brandITS; zmo=zinc+mineral oil; zcot=zinc+cottonseed oil; zcan=zinc+canolaoil.

In contrast, ITS formulations made with a triglyceride as the gel phase,rather than mineral oil, resulted in a dramatic (e.g., 10-fold) increasein the degree to which the residual soil is removed. See FIG. 5. Withoutbeing limited to a specific mechanism, it is believed that the improvedcleanability is a result of the increased chemical reactivity oftriglyceride as compared to the non-reactive mineral oil. In short, theester bond of the triglyceride material is readily hydrolyzed by thebasic cleaning agent (NaOH). As such, the soil is dispersed into theaqueous phase as a sodium salt of the corresponding fatty acids.

Example 4—Dairy Product Reactivity

Fluid milk samples (2% homogenized, vitamin D) were inoculated with 0.05wt % of each ITS treatment and stored at 45° F. for two weeks.Afterward, the samples were visually evaluated for signs of spoilageusing sensory methods using an expert panel (n=4). There were four ITStreatments evaluated, including “ORBESEAL”-brand ITS, Zn/mineral oil,Zn/canola oil, and a control sample. The panelists were asked to use adifference from control assessment. In short, the panelists were askedto evaluate the control sample, then rate the treated samples on flavorwith the control milk as a reference. Results were as follows:

There were no visual signs of protein destabilization whatsoever. Eachof the treated milk samples appeared undistinguishable from the control.Flavor assessments yielded similar results in that no significant flavordefects or attributes arose as a function of the presence or type of ITStreatment.

Cheese analysis is still in progress. Freshly made mozzarella andcheddar cheese samples were manufactured and the surfaces were treatedwith ITS, vacuum packaged, and are currently in an aging program. Todate, no deleterious signs or appearance defects are present.

Example 5—Yogurt Fermentation and Sensory Assessment

Three different yogurt fermentations were conducted in order toinvestigate a possible effect of the presence of the ZnO sealant or the“ORBESEAL”-brand ITS on the final sensory characteristics of thisfermented product. In previous experiments, it was determined that thepresence of such materials did not alter the drop in the pH of theproduct in a noticeably way. Hence, the following experiment wasperformed: 50 mL of pasteurized milk were place in three sterile 100 mLflasks, and inoculated with 0.5 mL of a suspension containingLactobacillus delbrukeii spp. bulgaricus (in Elliker's broth, previouslyincubated for 24 hour at 37° C.). 50 mg of “ORBESEAL”-brand ITS wasadded to one of the flasks and 50 mg of the ZnO sealant to other (65% inheavy mineral oil), the third flask was used as the control treatment.The flasks were incubated overnight at 37° C. and assessed fordifferences in the final pH, as well as flavor and aroma profiles.

Results: The final pH of the three fermented products was very similar,and ranged between 4.45 and 4.5. No differences in flavor and aromaprofiles were detected between the three treatments when tasting thefinal products.

Example 6—Density of ITS Formulations

The densities of 7 different zinc-based ITS formulations were determinedand compared to “ORBESEAL”-brand ITS. Aliquots (400 μL) of each of thesealant preparations were weighed using an analytical scale. Table 6summarizes the results obtained for each sample. The tabular entriescorrespond to the average of at least 10 measurements with correspondingstandard deviations. In general, the density of the suspensionsincreased when using higher amounts of ZnO. The performance criterionwas a range of 5-10% of the density of the original “ORBESEAL”-brandITS. This is a range of from about 1.817 to about 2.009 g/mL at the 5%level and about 1.722 to about 2.104 g/mL at the 10% level. All of thesamples tested fell within these parameters with the exception of the60% ZnO Canola Oil treatment at the 5% level.

TABLE 6 Densities of Different ITS Formulations. Product Density (g/mL)Orbeseal 1.913 ± 0.018 65% ZnO (Heavy Mineral Oil). 1.907 ± 0.023 65%ZnO (Light Mineral Oil). 1.902 ± 0.011 70% ZnO (Ligth Mineral Oil).1.915 ± 0.007 65% ZnO (Canola Oil). 1.990 ± 0.010 63% ZnO (Canola Oil).1.870 ± 0.029 60% ZnO (Canola Oil). 1.732 ± 0.036 65% ZnO (CottonseedOil). 1.937 ± 0.010

Example 7—Viscosities of ITS Formulations

The viscosities of newly prepared ITS comprising zinc oxide, aluminumstearate, and canola oil was determined, at several temperatures, usinga Brookfield Digital Viscometer Model DV-I Prime (Brookfield,Middleboro, Mass.) fitted with a S06 Spindle at 2.0 rpm. The effect ofthe temperature on the viscosity of these new ITS formulations can beobserved in FIG. 6. Data at lower temperatures (−15° C.) is in progress.

1. A method of forming a physical barrier in the teat canal of anon-human animal for prophylactic treatment of mammary disorders duringthe animal's dry period and simultaneously preventing black spot defectin dairy products made with milk from the animal, the method comprising:infusing an amount of a teat seal formulation into the teat canal of theanimal, wherein the teat seal formulation comprises a non-toxic metalsalt, dispersed in a gel phase comprising a glyceride, wherein theamount of the teat seal formulation infused is sufficient to form aphysical barrier to entry of microorganisms into the teat canal, andwherein the teat seal formulation does not cause black spot defect indiary products made with milk from the animal.
 2. The method of claim 1,comprising infusing a teat seal formulation that is devoid ofanti-infective agents.
 3. (canceled)
 4. The method of claim 1,comprising infusing a teat seal formulation that is devoid ofanti-infective agents and devoid of bismuth.
 5. The method of claim 1,comprising infusing a teat seal formulation comprising a bismuth-free,non-toxic metal salt in a base comprising triglyceride.
 6. The method ofclaim 1, comprising infusing a teat seal formulation comprising at leastabout 30% by weight of the non-toxic metal salt.
 7. The method of claim1, comprising infusing a teat seal formulation comprising about 50% toabout 75% by weight of the non-toxic metal salt.
 8. The method of claim1, comprising infusing a teat seal formulation comprising about 65% byweight of the non-toxic metal salt.
 9. The method of claim 1, whereinthe non-toxic metal salt is selected from the group consisting oftitanium salts, zinc salts, barium salts, and combinations thereof. 10.The method of claim 1, wherein the non-toxic metal salt is selected fromthe group consisting of titanium dioxide, zinc oxide, barium sulfate,and combinations thereof.
 11. The method of claim 1, wherein the gelbase comprises an oil selected from the group consisting of animal fats,canola oil, coconut oil, corn oil, cottonseed oil, linseed oil, palmoil, palm kernel oil, rapeseed oil, soya bean oil, sunflower oil, fishoil, algae oil, and combinations thereof.
 12. An intra-mammary teatsealant comprising, in combination: a gel base comprising a glyceride;and a non-toxic metal salt dispersed in the gel base.
 13. The teatsealant of claim 12, wherein the gel base comprises a triglyceride. 14.The teat sealant of claim 12, wherein the gel base comprises an oilselected from the group consisting of animal fats, canola oil, coconutoil, corn oil, cottonseed oil, linseed oil, palm oil, palm kernel oil,rapeseed oil, soya bean oil, sunflower oil, fish oil, algae oil, andcombinations thereof.
 15. The teat sealant of claim 12, wherein the teatsealant is devoid of anti-infective agents. 16-17. (canceled)
 18. Theteat sealant of claim 12, comprising at least about 30% by weight of thenon-toxic metal salt.
 19. The teat sealant of claim 12, comprising about50% to about 75% by weight of the non-toxic metal salt.
 20. The teatsealant of claim 12, comprising about 65% by weight of the non-toxicmetal salt.
 21. The teat sealant of claim 12, wherein the non-toxicmetal salt is selected from the group consisting of titanium salts, zincsalts, barium salts, and combinations thereof.
 22. The teat sealant ofclaim 12, wherein the non-toxic metal salt is selected from the groupconsisting of titanium dioxide, zinc oxide, barium sulfate, andcombinations thereof.
 23. In a method of forming a physical barrier inthe teat canal of a non-human animal for prophylactic treatment ofmammary disorders during the animal's dry period, the method comprisingthe step of infusing a seal formulation into the teat canal of theanimal, an improvement comprising: infusing a teat seal formulation asrecited in claim 12.